Ecological and Evolutionary Physiology of the Stress Response and Stress Proteins

应激反应和应激蛋白的生态学和进化生理学

• 批准号: 0316627
• 负责人: Martin Feder
• 金额: $ 58.12万
• 依托单位: University of Chicago
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0316627&HistoricalAwards=false
• 关键词: Ecological; Evolutionary; Physiology; Stress; Response

Broad goals: The proposed activity continues a research program whose goal is to understand the origin and maintenance of complex physiological and biochemical mechanisms that enable organisms to persist in challenging environments. This program uses as a model system the heat-shock protein Hsp70, which minimizes stress-induced damage of other proteins, and the genes that encode it in the fruit fly, Drosophila. The principal remaining unanswered questions are: What is the genetic basis for the variation in Hsp70 levels among natural populations, and how does this variation arise? Prior work suggests that transposable elements, DNA sequences that can move from gene to gene, are the answer to these questions. The major goal of the research is to test this hypothesis.Specific aims: First the research will determine the frequency with which transposable elements actually insert in the genes encoding heat-shock proteins, and compare this frequency with that for other genes that either do or do not resemble heat-shock genes in their structure, organization, and regulation. In so doing, the research will discover numerous instances of transposable elements naturally inserting into heat-shock genes. Next, the research will use these natural mutants to examine whether the insertions actually have the expected effect on heat-shock genes: reduction in messenger RNA, heat-shock protein, and ability to withstand stress. Finally, the researchers will undertake "laboratory evolution", in which fruit fly strains compete in environments that ought to favor retention or elimination of individuals with and without transposable elements in their heat-shock genes. This final experiment would test whether transposable elements in the heat-shock genes actually have their expected effect on individual fitness.Approaches: (a) Bioinformatics, the analysis of pre-existing information to predict genes' properties. (b) High-throughput screening, the simultaneous and rapid analysis of many genes in many populations to detect genetic events. This will use "universal fast walking", a new technique for cloning genes. (c) Transcriptional profiling, the quantification of messenger RNAs produced by specific genes when they are expressed. This profiling would use two techniques, the fusion of the heat-shock genes' regulatory region with a reporter protein that emits light when the gene is expressed, and quantitative real-time PCR, which measures messenger RNA by quantifying its accumulation in a chemical reaction. (d) Laboratory evolution, as described above.Significance: The proposed research is distinctive in that it will elucidate a mechanism that apparently has repeatedly resulted in the decreased expression of a protein, with adaptive consequences. Because of extensive background work on the genes/protein in question, it can rigorously assign significance to both regulatory mutations and variation in the level of the protein they regulate. The proposed research has the potential to become a landmark study in which all of the often-missing links among evolutionary generation of variation, gene sequence, cellular function, organismal function, fitness, gene frequency in populations, and response to natural selection are explained.

总体目标：拟议的活动继续一项研究计划，其目标是了解使生物体能够在具有挑战性的环境中持续存在的复杂生理和生化机制的起源和维持。 该计划使用热休克蛋白Hsp 70作为模型系统，该蛋白最大限度地减少了其他蛋白质的应激诱导损伤，以及果蝇中编码它的基因。 剩下的主要问题是：什么是在自然人群中的HSP 70水平的变化的遗传基础，以及这种变化是如何产生的？ 先前的工作表明，转座因子，可以从基因转移到基因的DNA序列，是这些问题的答案。 本研究的主要目的是验证这一假设。具体目的：首先，研究转座因子实际插入编码热休克蛋白的基因中的频率，并将此频率与其他在结构、组织和调控方面与热休克基因相似或不相似的基因进行比较。 在这样做的过程中，这项研究将发现许多转座因子自然插入热休克基因的例子。 接下来，研究人员将使用这些天然突变体来检查插入是否真的对热休克基因产生了预期的影响：信使RNA、热休克蛋白和承受压力的能力减少。 最后，研究人员将进行“实验室进化”，即果蝇品系在环境中竞争，这种环境应该有利于保留或消除热休克基因中有或没有转座因子的个体。 这最后一个实验将测试热休克基因中的转座因子是否真的对个体适应性产生预期的影响。方法：（a）生物信息学，分析预先存在的信息以预测基因的特性。 (b)高通量筛选，同时快速分析多个群体中的多个基因以检测遗传事件。 这将使用“通用快速行走”，一种克隆基因的新技术。 (c)转录谱分析，定量特定基因表达时产生的信使RNA。 这种分析将使用两种技术，一种是将热休克基因的调控区与一种报告蛋白融合，当基因表达时，报告蛋白会发光，另一种是定量实时PCR，通过量化信使RNA在化学反应中的积累来测量信使RNA。 (d)实验室进化，如上所述。意义：拟议的研究的独特之处在于，它将阐明一种机制，该机制显然反复导致蛋白质表达减少，并产生适应性后果。 由于对相关基因/蛋白质的广泛背景研究，它可以严格地将重要性分配给调控突变和它们调控的蛋白质水平的变化。 拟议的研究有可能成为一个具有里程碑意义的研究，其中解释了进化产生的变异，基因序列，细胞功能，有机体功能，健身，种群中的基因频率和对自然选择的反应之间的所有经常缺失的联系。